Activation of the MMTV LTR promoter by steroid hormones proceeds through the assembly of a transcription initiation complex composed of factors NF1/CTF, Octamer binding protein, and TFIID. This complex is assembled in vivo on two (A and B) of the highly positioned nucleosomes on the LTR. A major issue under investigation has been whether the highly organized nucleoprotein structure is mechanistically involved in transcription activation. We have found that loading of NF1 occurs constitutively on transiently transfected DNA, whereas its binding in stable chromatin is hormone dependent. Thus, recruitment of some factors (NF1) requires a chromatin modification mechanism, whereas other factors (Oct-1) probably interact directly with receptor or adaptor proteins. It was also discovered that a progesterone receptor transiently introduced into mouse cells is incapable of activating stable, replicated MMTV promotor chromatin, while transiently introduced, LTR reporter is highly responsive. These results demonstrate that factor access to DNA in stable chromatin is specifically and selectively modulated by nucleoprotein structure.